Approximately 350,000 anterior cruciate ligament (ACL) reconstruction surgeries are performed each year, and billions of dollars are spent on the acute care costs involved in this procedure. The most common ACL replacement strategies involve grafts (allografts from cadavers or autografts of the patients' own patellar or hamstring tendons), although newer, more experimental treatments use engineered tissues that typically involve seeding cells on a polymeric scaffold. In these procedures, the graft or engineered tissue never fully integrates with the bone tunnel created during surgery and the joint never recovers pre-surgical level biomechanics. Not surprisingly, more than half of the patients who have undergone this procedure develop early-onset osteoarthritis (OA). With acute ACL injuries becoming increasingly prevalent in children, the number of young adults developing OA is increasing each year. The Larkin and Arruda laboratories have spent the last several years engineering an ACL replacement construct generated from bone marrow-derived stromal cells (BMSCs) that exhibits the structural and functional interface characteristics of native ACL when transplanted in vivo. The construct is composed of an engineered ligament with engineered bone at each end; it fully integrates into the recipient bone and differentiates in vivo to form a mechanically appropriate and biologically matched interface between the two tissues. Work from these laboratories demonstrates many advantages to this strategy and evidence in a large animal model supports its superiority for replacement therapy. One critical concern regarding the use of this novel and innovative therapy in humans, however, is the safety of use of stem/precursor cell-derived tissue in patients. It is imperative to demonstrate that the BMSCs used for the generation of these constructs pose no long-term threat after transplantation. The main concern in the field is the ability to ascertain that no undifferentiated cells persist in the transplanted construct that might later lead to aberrant cellular behavior such as cancer. In collaboration with Dr. Wellik's laboratory, a recent examination of the fate of the BMSC-derived BLB construct led to the surprising discovery that within several months after implantation of the BLB, donor construct cells are replaced entirely by recipient cells and donor-derived cells are no longer present. Thus, by transplanting a developmentally immature, exogenous live tissue template for replacement, adult recipient cells are induced to fully regenerate a viable and mechanically appropriate replacement ligament! This proposal seeks to confirm these preliminary results and initiate exploration into the mechanisms of this remarkable regenerative process, with the longer-term goal of translating this innovative therapy into humans and vastly improving the outcomes of acute ACL and potentially other joint and connective tissue injuries. PUBLIC HEALTH RELEVANCE: Billions of dollars are spent each year on anterior cruciate ligament (ACL) reconstructions after acute knee injury and more than half of patients who undergo this procedure develop early- onset osteoarthritis. New technology developed by this research team uses bone marrow stromal cell-derived, living bone-ligament-bone templates to replace the injured ACL. Transplanted tissue does not survive long-term in the recipient (patient), but induces the regeneration of a new ligament made entirely of the patient's own cells! The regenerating ACL integrates into bone and forms a viable and mechanically appropriate replacement ligament. If these preliminary results are confirmed, we will be one large step closer to translating these results into a vastly improved human therapy. Further, understanding the mechanisms by which this regeneration occurs will open new avenues in the regeneration of musculoskeletal tissue.